Methods of Making Di-Tagged DNA Libraries from DNA or RNA Using Double-tagged Oligonucleotides

ABSTRACT

Disclosed are methods, compositions and kits related to making double-tagged DNA libraries from RNA/DNA samples. A double-tagged oligonucleotide (DTO) is employed to efficiently add two different tags to ends of DNAs to make a double-tagged DNA libraries. Also disclosed are methods to make mate pair libraries using the double-tagged oligonucleotide, and methods to make double-tagged single stranded DNA. The double-tagged DNA libraries of the invention are ready to be used on next generation sequencing machines.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.13/466,610 filed May 8, 2012, which claims the benefit of U.S.provisional patent application No. 61/483,710, filed May 8, 2011, thecontents of both applications are incorporated by reference herein.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention provides methods, compositions and kits related tomaking double-tagged DNA libraries using a double-taggedoligonucleotide. It particularly relates to methods, compositions andkits for making di-tagged dsDNA libraries suitable for high throughputsequencing from DNA and RNA samples.

2. Description of the Related Art

Next Generation Sequencing (NGS) is a high-throughput sequencingtechnology that performs thousands or millions of sequencing inparallel. The NGS technology enables researchers to answer fundamentalbiological questions at a genomic scale and has great potential inmedical applications. The first step to apply NGS technologies isconstructing DNA libraries of random DNA fragments generated fromDNA/RNA samples, having different sequence tags attached at both ends.Preparation of high quality di-tagged DNA library is critical forsuccessful sequencing.

As Next Generation Sequencing technologies evolve and advance, there aregrowing demands for PCR free library preparation, Mate Pair librarypreparation and single stranded library preparation to serve researcherswith specific project needs. The challenges of PCR free librarypreparation is the ligation step: current method involves ligating twodifferent sequence tags to blunt ended DNA fragments using a DNA ligase.Due to random ligation of different tags to ends of DNA fragments,ligation products include DNA sequences with only one tag, with the sametags, or with two different tags. PCR amplification is needed toselectively amplify the DNA fragments having different tags. An improvedligation method uses one “Y” shaped tag having a non-complementary outerportion and a complementary inner portion. Two strands of thenon-complementary outer portion have different sequence tags, and twostrands of the complementary inner portion anneal to each other to forma dsDNA that can be ligated to dsDNA fragments. Once a DNA is ligated totwo “Y” shaped tags, each DNA strand will be ligated to differentsequence tags of the non-complementary outer portion. However, selectivePCR amplification is still needed since this method does not exclude thepossibility that DNA is ligated to only one “Y” shaped tag.

Using a double-tagged oligonucleotide, the present invention provides amethod of making a di-tagged DNA without the need of performing a PCR,therefore eliminating the sequence bias caused by PCR.

A mate pair DNA refers to a DNA sequence comprising two DNA segmentsoriginally located long distance apart in the genome. A mate pairlibrary is comprised of mate pair DNA sequences with two differentsequence tags attached at the ends. Mate pair sequencing is useful formany applications such as genomic sequence assembly, assessment ofgenomic rearrangement, and assembly of repetitive sequences. To make amate pair library, end sequences of a large DNA fragment are connectedtogether and attached to two different sequence tags. Current techniquesfor preparing mate pair DNA libraries involve fragmenting genomic DNAinto large fragments, performing end repairs, labeling ends of large DNAfragments with biotin, self-ligation of biotinylated ends, randomfragmentation to generate smaller DNA fragments, isolation ofbiotinylated fragments, performing end repairs, and ligation of twodifferent tags to the DNA fragments. This procedure comprises manyenzymatic and cleaning steps leading to low yield of mate pair DNAs. Thelow ligation efficiency of biotinylated nucleotides further limits theapplication of this method.

Using a double-tagged oligonucleotide, the present invention provides amethod of making Mate-Pair DNA libraries with less enzyme reactions andless purification steps, resulting in simpler workflow and betterrecovery.

Single stranded DNA library has many applications such as DNAmethylation analysis and hybridization based target capture. Existingmethod for generating single stranded DNA involves ligation of anadaptor to a dsDNA molecule, and uses only one primer to linearlyamplify one single strand. One of the major drawbacks for this method isthe sequence bias introduced by PCR cycles.

Using a double-tagged oligonucleotide, the present invention provides asimple PCR-free method to generate single stranded DNA library fromdsDNA, eliminating the sequence bias caused by PCR. The resultinglibrary would well reflect the complexity and composition of the DNAsequences of the starting materials.

RNA sequencing (RNA Seq) uses high throughput sequencing technology forquantifying and mapping transcriptomes, enabling rapid profiling anddeep investigation of transcriptional activities. The first step ofRNASeq is to convert a population of RNA molecules into a library of DNAmolecules with different tags attached to both ends, which can then besequenced in a high throughput manner. The conventional method forpreparing tagged DNAs from RNAs involves reverse transcription togenerate first and second complementary DNA (cDNA) strands, DNAfragmentation, DNA ends repairing, adaptor ligation, and PCRamplification. These procedures include multiple enzyme reactions andbuffer exchanges between different enzyme reactions, resulting insignificant loss of starting materials. Sequence-specific bias may alsobe introduced during ligation and amplification steps.

Using a single stranded, double-tagged oligonucleotide, the presentinvention provides a method of making RNA libraries without synthesizingsecond strand cDNA, resulting not only simpler work flow, but alsoprovides directional information on the RNA Transcripts. Moreimportantly, it offers a library preparation solution to RNA samples ofextremely small amounts, such as applications for CLIP Seq.

SUMMARY OF THE INVENTION

The present invention pertains to methods, compositions and kits relatedto making di-tagged DNA libraries from RNA/DNA sequences, which aresuitable for using in high throughput sequencing. The present inventionemploys a Double-Tagged Oligonucleotide (DTO) to make di-tagged DNAs,allowing efficient addition of different tags to both ends of a DNAsequence.

The present invention provides a DTO that sequentially comprises apre-selected sequence tag A, a linker, and a pre-selected sequence tagB. The sequence tags A and B are preselected to match sequence tagsspecific for sequencing platforms. The DTO can be a double stranded DNAoligo used for ligation with two ends of double stranded DNAs to form acircular molecule. The double stranded DTO may have nick(s), gap(s),modified nucleotides, abasic nucleotides, or other chemical moieties inits linker region. The DTO can be a single stranded DNA oligo used as aprimer for synthesizing a complementary DNA from a RNA or DNA sequence.The single stranded DTO further comprises a priming sequence at its 3′end.

The linker of the DTO comprises a natural or non-natural nucleotidesequence located between sequence tags A and B, that provides a breakingsite or a stopping sequence. A breaking site refers to a region in a DTOthat is susceptible to photo, enzymatic, or chemical cleavage. Thebreaking site may comprise a single-stranded DNA/RNA sequence or adouble-stranded DNA sequence with modified nucleotides and/or othernon-nucleotide chemical moieties. A stopping sequence refers to modifiednucleotides or non-nucleotide chemical moieties that can stop elongationof a DNA during a DNA polymerization reaction.

In some embodiment, the DTO further comprises a priming sequence at its3′ end. The priming sequence is a sequence or a mixture of sequencesthat is complementary to part of a RNA/DNA sequence and used as a primerfor synthesizing a complementary DNA (cDNA). The priming sequence, forexample, can be a mixture of random hexamers for annealing to anycomplementary location of a RNA/DNA sequence or a oligo(dT) forannealing to a 3′ end of any RNA with a polyadenine (poly(A)) tail. Theterm “complementary DNA” or “cDNA” used herein refers to asingle-stranded DNA sequence that is synthesized from and iscomplementary to a RNA or DNA template. A cDNA complementary to a RNAtemplate can be synthesized using a reverse transcription reaction, anda cDNA complementary to a DNA template can be synthesized using a DNApolymerization reaction.

In one embodiment, the present invention provides a method of making adi-tagged DNA library, comprising the steps of: a) providing adouble-tagged oligonucleotide (DTO), sequentially comprising a sequencetag A, a linker, and a sequence tag B; b) providing a DNA or RNAfragment; c) connecting the DTO to the DNA fragment or a cDNA fragmentgenerated from the DNA or RNA fragment to form a circular DNA-DTOmolecule; e) generating a linear DNA from the circular DNA-DTO moleculewith sequence tag A and tag B at its ends. The collection of such linearDNAs with sequence tag A and tag B forms a di-tagged DNA library. Insome embodiment, the linear DNA fragments or free DTOs are digested byexonucleases so that they can be easily separated from the circularDNA-DTO molecules.

The linker of the DTO comprises a stopping sequence or a breaking site.The stopping sequence comprises modified nucleotides or chemicalmoieties that can stop a DNA polymerization reaction. The stoppingsequence may comprise modified nucleotide analogues that cannot formbase pairs with natural nucleotides; modified nucleotide analogues thatcan form base pairs with natural nucleotides, but are structurallyincompatible with polymerases; and chemical moieties with littlestructural similarities to nucleotides that can function to stop DNApolymerization. The breaking site comprises a special region of the DTOthat is susceptible to photo, enzymatic or chemical cleavage. The DTOmolecule can be a single stranded or a double stranded DNA, which can beconnected to DNA fragments or cDNA fragments via ligation, reversetranscription or DNA polymerization. There are two methods forgenerating a linear DNA from a circular DNA-DTO molecule. If there is abreaking site within the DTO sequence, linear DNAs with sequence tag Aand tag B can be generated by breaking at the breaking site.Alternatively, using a primer pair corresponding to sequence tag A andtag B, a PCR is performed to generate a di-tagged linear DNA from thecircular DNA-DTO molecule.

In one embodiment, the invention provides a method for making adi-tagged DNA from double stranded DNA sequences. The method comprisesthe following steps: a, ligate two ends of a double stranded DNAfragment to a double-stranded DTO with a breaking site to make acircular dsDNA; b, use an appropriate method to cleave the circularsequence at the breaking site to generate a linear DNA sequence with twodifferent sequence tags (tag A and tag B) at each end of the sequence.In some embodiment, the breaking site comprises uracil nucleotides, asingle stranded DNA, a single-stranded RNA, or a double stranded RNAsequence. In other embodiment, the breaking site comprises aphoto-cleavable nucleotide spacer or nucleotide analogs susceptible tochemical cleavage.

In some embodiment, the invention provides a method of making adi-tagged DNA comprises the steps of the following: a, ligate adouble-stranded DTO to DNA fragments to form a circular DNA-DTO product;b, use sequence tag A and B as PCR primers to amplify the DNA fragmentswith sequence tag A and B at both ends.

In one embodiment, the present invention provides a simple and highlyefficient method for making mate pair DNA libraries. The methodcomprises the following steps: a, ligate a double stranded DTO to largeDNA fragments to generate large circular DNAs; b, randomly fragmentlarge circular DNAs to small DNA fragments; c, perform end repair of thesmall DNA fragments to make ready for ligation; d, self-ligate the smallDNA fragments to generate small circular DNA; e, perform PCR with thesmall circular DNA to generate a linear DNA with tags A and B, whichcontains sequences of both ends from a same large DNA fragment. In someembodiment, the linear DTOs and DNA fragments are digested byexonuclease treatment and are removed from the closed-circular DNA-DTOmolecules. In some embodiment, the DTO comprises a breaking site in itslinker region. The small circular DNA is cleaved at the breaking site togenerate a linear DNA with two sequence tags at the ends. A PCR isperformed on the linear DNA to select DNA sequences with two sequencetags. In some embodiment, the DTO comprises a stopping sequence in itslinker region.

In some embodiment, the present invention provides a method of making amate pair DNA libraries from large DNA fragments using a nicked DTO. Themethod comprises the steps of the following: a, ligate both ends of alarge DNA fragment to a double-tagged oligonucleotide to generate alarge circular DNA, wherein the double-tagged oligonucleotide has a nicksite on each of the opposite strand; b, perform a nick translation toelongate the 3′ end of each nick site; c, break the large circular DNAat the nick sites to generate a smaller linear DNA with thedouble-tagged oligonucleotide; d, end repair the smaller linear DNA tomake it ligation ready; e, self-ligate the smaller linear DNA to form asmall circular DNA that contains end sequences of a large DNA fragmentand double-tagged oligonucleotide; f, perform PCR with the smallcircular DNA to generate a linear DNA with tags A and B. In someembodiment, the linear DTOs and DNA fragments are digested byexonuclease treatment and are removed from the circular DNA-DTOmolecules. In some embodiment, the DTO comprises a breaking site in itslinker region. The small circular DNA is cleaved at the breaking site togenerate a linear DNA with two sequence tags at the ends. A PCR isperformed on the linear DNA to select DNA sequences with two sequencetags.

The invention provides a method for making a di-tagged DNA from a RNAsequence using a single stranded DTO with a priming sequence. The methodcomprises the following steps: a, annealing the single stranded DTO tothe RNA sequence; b, extending the 3′ end of the DTO using a reversetranscriptase to make a cDNA-DTO molecule; c, ligating the 3′ and 5′ends of the cDNA-DTO to generate a circular cDNA-DTO; d, thecircularized cDNA-DTO is further amplified by PCR to generate a lineardsDNA with sequence tag A and tag B at its ends. In some embodiment, asingle stranded DNA specific nuclease is used to digest free singlestranded DTOs before self-ligation of cDNA-DTO molecule. In oneembodiment, the priming sequence of the DTO comprises a random hexamer.In another embodiment, a poly(A) tail is added to the 3′ end of RNAmolecules by a poly(A) polymerase and a DTO with a poly(dT) is used as aprimer for synthesizing a cDNA. In some embodiment, biotinylated AMPsare incorporated into the poly(A) tail of RNA molecules during thepolyadenylation process. The biotin-RNA can be used to separatecDNA/biotin-RNA hybrids from unbound DTOs. In some embodiment,biotinylated dNTP can be used to incorporate biotinylated nucleotideinto the cDNA during the reverse transcription reaction. The biotin-cDNAcan be separated from unbound DTOs using Streptavidin magnetic beads. Insome embodiment, the DTO comprises a breaking site, and the linear DNAis generated from the circular cDNA-DTO by breaking at the breakingsite.

In some embodiment, the invention provides a method for making adi-tagged DNA from a single-stranded DNA sequence using asingle-stranded DTO with a priming sequence. The method comprises thefollowing steps: a, annealing the single stranded DTO to the DNAsequence; b, extending the 3′ end of the DTO using a DNA polymerase tomake a cDNA-DTO; c, self-ligating the 3′ and 5′ ends of the cDNA-DTO toform a circular cDNA-DTO. The circularized cDNA can be amplified by PCRto generate a linear dsDNA with sequence tags A and B. In oneembodiment, the priming sequence of the DTO comprises a random hexamer.In another embodiment, a poly(dA) tail is added to the 3′ end of the DNAmolecules by a terminal transferase and a DTO with a poly(dT) is used asa primer for synthesizing a cDNA. Biotinylated dAMPs is incorporatedinto the poly(dA) tail of DNA molecules using a terminal transferase.The biotin-poly(dA) tail can be used to separate cDNA/biotin-DNA hybridsfrom unbound DTOs.

In some embodiment, the present invention provides a method of making adi-tagged single-stranded DNA from a double-stranded DNA. The methodcomprises the steps of the following: a, ligate a double-stranded DNA toa double-tagged oligonucleotide to generate a circular dsDNA, whereinthe double-tagged oligonucleotide has a single nick or a gap; b, removethe nicked/gapped strand using exonucleases to generate a circularsingle-stranded DNA; c, break the circular single-stranded DNA at thebreaking site to generate a linear di-tagged single-stranded DNA. Inanother embodiment, the double-tagged oligonucleotide comprises astopping sequence. The DTO may or may not have a nick or gap. After theDTO and the dsDNA sequence are ligated to form a circular DNA-DTO, usingone primer annealing to sequence tag A or B, single-stranded DNA can beamplified by a linear PCR. The stopping sequence ensures the generationof single-stranded DNAs with two sequence tags at the ends, preventinggeneration of rolling cycle products by PCR amplification.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. A scheme of double-tagged oligonucleotides.

FIG. 2. A schematic illustration of a PCR-free method for adding twotags to a dsDNA using a DTO. 2A, method for making di-tagged DNAs usinga DTO with uracil nucleotides. 2B, method for making di-tagged DNAsusing a DTO with a single stranded gap.

FIG. 3. A schematic illustration of making di-tagged mate pair DNAsusing a dsDTO.

FIG. 4. A schematic illustration of making di-tagged mate pair DNAsusing a nicked dsDTO.

FIG. 5. A schematic illustration of making double-tagged DNA moleculesfrom RNA molecules using random priming sequences.

FIG. 6. A schematic illustration of making double-tagged DNA moleculesfrom biotinylated RNA molecules.

FIG. 7. Shows that DTOs prevent polymerase from reading through thestopping sequence.

A DTO with a stopping sequence of five abasic deoxyribonucleotides and aregular oligonucleotide of the same length were used as templates forstandard PCR amplification using the same forward and backward primerpair. The DTO prohibited polymerization by DNA polymerase across thestopping sequence, resulting in very low production of PCR product. Theregular oligonucleotide produced abundant PCR product of the expectedsize. DNA marker sizes are 25, 50, 100, 200, 300, 400, 500, 600, 700,800, 900, 1000 bp.

FIG. 8. Shows generation of double-tagged DNA using a DTO.

A synthesized 70 by DTO has, from 5′ to 3′, a sequence A tag, fiveabasic deoxyribonucleotides, a sequence B tag, and a poly(dT) tail+NV. Asynthesized 23 bp RNA oligo was polyadenylated using PolyA polymerase.The polyadenylated RNA oligo was purified, reverse transcribed with DTOoligo using M-MLV reverse transcrptase (Clonetech, Mountain View,Calif.) and circularized using CircLigase II (EPICENTRE, Madison, Wis.).The circularized cDNA product was amplified with 30 PCR cycles usingprimers corresponding to sequence tags A and B. Part of the PCR productwas digested with Msl I which only cuts at the ligation junction of cDNAand 5′ DTO. The size of the PCR products of DTO self-ligation andDTO-cDNA ligation are 70 bp and 93 bp, respectively. The 93 bp PCRproduct (upper band) disappeared after Msl I digestion, indicating thatthe disappearing product is the PCR product of cDNA and 5′ DTA oligoligation.

FIG. 9. A schematic illustration of making a di-tagged single strandedDNA from a dsDNA.

FIG. 10. Structure of nucleotide spacers

FIG. 11. Structure of nucleotide analogs

DETAILED DESCRIPTION

High-throughput sequencing technologies requires converting sequencesfrom RNA or DNA starting materials into random small DNA fragments withdifferent sequence tags attached at both ends. Different sequencingplatforms require different sequence tags to be added to DNA fragments.The present invention employs a DTO, sequentially comprising apre-selected sequence tag A, a linker, and a pre-selected sequence tagB. The sequence tags A and B are preselected to match sequencingplatform specific sequencing tags, allowing resulting di-tagged DNAsready to be sequenced on any sequencing platform of choice. The linkerprovides a breaking site or a stopping sequence.

The use of the DTO is advantageous in multiple respects. The DTOprovides two pre-selected sequence tags (tag A and tag B) in a singlemolecule that enables highly efficient addition of two sequence tags toa cDNA via inter and intra-molecular ligations. Using self-ligation forlinking a sequence tag is especially effective for small inserts (e.g.siRNA and snRNA) or RNA/DNA samples with very low concentrations.

For single stranded RNA or DNA, the use of the single stranded DTOsleads to differential tagging of the 5′ and the 3′ ends of a cDNAsequence, which maintains the information about the strand direction ofthe RNA/DNA sequence of origin. For example, using a DTO (from 5′ to 3′,a sequence tag A, a stopping sequence, a sequence tag B, and a primingsequence) as a primer for synthesizing a cDNA, the cDNA is connected tothe sequence tag B on its 5′ end. The sequence tag A is connected to the3′ end of the cDNA by way of self-ligation. Using a DTO to incorporatedifferent sequence tags into different ends of each cDNA sequenceensures that the strand of origin of the starting RNA/DNA can beascertained.

The stopping sequence in the DTO provides an “autostop” function toprevent DNA polymerase from further extending the 3′ end of asynthesizing nucleic acid. The stopping sequence comprises non-naturalnucleotide analogues or other chemical moieties that function as a stopcode for DNA polymerase. The stopping sequence prevents rolling circleamplification during PCR reactions, which avoids introduction ofsequence bias due to differences in rolling circle efficiency. Theefficiency of forming rolling circles is different for different DNAsequences depending on the size and composition of the molecules.Elimination of rolling circle amplification by the stopping sequence isadvantageous by eliminating sequence bias introduced in this respect.The use of stopping sequence also provides a cleaner background. Thebreaking site provide a simple way to generate linear DNAs from circularDNAs.

The present invention provides a DTO having a sequence tag A, a linker,a sequence tag B. The sequence tags A and B are preselected to matchsequence tags specific for sequencing platforms. The DTO can be a doublestranded DNA oligo used for ligation with two ends of double strandedDNAs to form a circular molecule. The double stranded DTO may havenick(s), gap(s), modified nucleotides, abasic nucleotides, or otherchemical moieties. The DTO can be a single stranded DNA oligo used as aprimer for synthesizing a complementary DNA from a RNA or DNA sequence.The single stranded DTO further comprises a priming sequence at its 3′end.

The linker of a DTO comprises a natural or non-natural nucleotidesequence, or non-nucleotide chemical moieties located between sequencetags A and B. The function of the linker is to provide a breaking siteor a stopping sequence. A breaking site of a DTO refers a special regionof the DTO that is susceptible to photo, enzymatic or chemical cleavage.The breaking site may comprise a single-stranded DNA/RNA sequence, adouble-stranded RNA, or a double-stranded DNA sequence with modifiednucleotides and/or other non-nucleotide chemical moieties. In someembodiment, the breaking site comprises a single-stranded DNA sequencein a double stranded DTO that provides a cleavage site for S1 nucleasewithin the dsDTO. In some embodiment, the breaking site comprises uracilnucleotides, which can be converted to a baseless nucleotide by UracilDNA Glycosylases (UDG) and be subjected to cleavage by AP endonucleases.

In some embodiment, the breaking site comprises modified nucleotidesthat are susceptible to chemical cleavage. For example, 5-hydroxy-dCTP,5-hydroxy-dUTP, 7-Deaza-7-nitro-dATP, 7-Deaza-7-nitro-dGTP (see FIG. 11)are modified nucleotides that form normal base pairing and are subjectedto chemical cleavage by KMnO4 and pyrrolidine treatment (Wolf, J L, ProcNatl Acad Sci U S A., 2002 99(17):11073-8). A breaking site canincorporate at least one of these modified nucleotides into the each oneof its strands.

In some embodiment, the breaking site comprises a photo-cleavablechemical moiety which can be cleaved by exposure to UV light. Thephoto-cleavable chemical moiety can be, for example, a 9 atomphoto-cleavable nucleotide spacer (PC spacer, see FIG. 10) commerciallyavailable from Integrated DNA Technologies (Coralville, Iowa). The PCspacer can be incorporated into the position between sequence tag A andtag B to serve as a breaking site. Photo cleavage of a PC spacerreleases an oligo nucleotide with a 5′ phosphate.

The stopping sequence of the DTO comprises, instead of four naturalnucleotides, chemical moieties that cannot effectively serve astemplates for polymerases and function to stop enzymatic nucleic acidpolymerization. Such chemical moieties include, but not limited to,modified nucleotide analogues that cannot form base pairs with naturalnucleotides, modified nucleotide analogues that can form base pairs withnatural nucleotides, but are structurally incompatible with polymerases,and chemical moieties with little structural similarities to nucleotidesthat can function to stop DNA polymerization. As the stopping sequencemay comprise modified nucleotides or chemical moieties that do not formbase pairing, the stopping sequence within a double stranded DTO maycreate a gap in the dsDTO. The 5′ and 3′ end nucleotides of the stoppingsequence gap may be modified to confer nuclease resistance. For example,the 5′ and 3′ hydroxyl group can be replaced with a —NH₂ group so thatthey are resistant to nuclease cleavage.

In some embodiment, the stopping sequence comprises one or morenucleotide analogs with bases that are sterically or electronicallyincompatible with the active sites of polymerases, which are effectiveat stopping nucleic acid polymerization at the site of such analogs. Forexample, 4-methylidole β-nucleoside, α-naphthalene nucleoside, andα-pyrene nucleoside, which are non-polar nucleotide analogs that pairspoorly with natural nucleotide bases, can be incorporated into the DTOsas the stopping sequence. The method to synthesize and incorporate thesemodified nucleotide analogs into DTA oligo was described in Moran etal., Nucleic Acids Research, 1996, vol. 24, No. 11, page 2044-2052,which is incorporated by reference herein. Incorporation of one of thesemodified nucleotide analogs may have different efficiency as to stoppingenzymatic polymerization. Incorporation of at least 2, 3, 4, 5 or moremodified nucleotide analogs can greatly enhance the autostop functionand lead to complete stop of nucleic acid polymerization by polymerasesbeyond the stopping sequence.

In some embodiment, the stopping sequence comprises ribose-phosphate ordeoxyribose-phosphate backbone without nucleobases. It is reported thatsome DNA polymerases can “read through” one abasic nucleotide andintegrate a natural nucleotide on the opposite strand. Abasicnucleotides are more effective at inhibiting DNA polymerization bypolymerases with 3′ to 5′ proofreading activities as proofreadingpolymerases will recognize the erroneous basepairing with abasicnucleotides and remove the erroneous nucleotide from the 3′ end (Gal, etal. Analytical Biochemistry, 2000, 282, 156-158). Incorporation of morethan one (e.g. 2, 3, 4, 5, or more) abasic nucleotides into the stoppingsequence can effectively stall the polymerization by most polymerases.In FIG. 4, incorporation of five abasic nucleotides in a DTO effectivelyprevented DNA polymerase from reading through the stopping sequence.

In some embodiment, the stopping sequence comprises nucleotide analogsor mimics that can form base pairs with natural nucleotides, but cannotserve as templates for polymerases. It is contemplated that nucleotideanalogs with a bulky group on its nucleobase, which may maintain thebase pairing ability with natural nucleotides, but cannot fit into theactive site of DNA polymerases, can also be used in the stoppingsequences.

In another embodiment, the stopping sequence comprises internal spacersof different lengths between sequence tags A and B. Chemical moietiesthat completely lack structural similarity to natural nucleosides cannotbe recognized by polymerases or used as templates for nucleic acidpolymerization. When incorporated into DTOs, such chemical moieties canserve as effective stop codes for polymerases. For example, commerciallyavailable nucleotide spacers such as C3 spacer phosphoramidite, 9-atomtriethylene glycol spacer, and 18-atom hexa-ethyleneglycol spacer(Integrated DNA Technologies, Coralville, Iowa) (see FIG. 10), can beincorporated into DTOs as the stopping sequence to block nucleic acidpolymerization by DNA polymerases (Brukner et al. AnalyticalBiochemistry, 2005, 339, 345-347). In addition, multiple nucleotidespacers can be inserted between sequence tag A and B to introduce alonger spacer arm, which could more efficiently block DNApolymerization.

In some embodiment, the stopping sequence comprises a single strandedRNA or a double stranded RNA. Most DNA polymerases either cannot use RNAas template or does so at very low efficiency. Incorporating multipleribonucleotides into a DTO sequence can effectively prevent extendingDNA polymerization across the RNA region.

Further, the DTO may comprise a capture domain that allows capturing ofthe DTO. The term “capture domain” refers to a structure or a moietyincorporated into a nucleic acid sequence that allows the separation ofthe capture domain containing nucleic acid sequence and any specificallybound nucleic acids from the rest of nucleic acid populations. Thecapture domain may comprise an affinity binding group which allows thecapture of the capture domain containing nucleic acid by affinitybinding to its binding partner, or a cross-linking moiety that iscapable of photochemically or chemically forming a covalent bond toother immobilized substrate. Methods to separate nucleic acids byaffinity binding are well known to those of ordinary skill in the art.Non-limiting examples of the separation methods include using physicalseparation, ligand-receptor binding, antigen-antibody association, orcomplementary nucleic acid pairing. In some embodiment of the invention,a biotin moiety is incorporated into the DTO as a capture domain. Thebiotin-containing DTO can be separated by binding to immobilizedstreptavidin or avidin (e.g. streptavidin-coated magnetic beads oravidin-coated magnetic beads).

In some embodiment, the double-tagged oligonucleotide of the presentinvention further comprises a priming sequence. The priming sequencecomprises a sequence or a mixture of sequences that is complementary topart of a RNA/DNA fragment and used as a primer for synthesizing acomplementary DNA (cDNA). The priming sequence, for example, can be amixture of random hexamers for annealing to any complementary locationof a RNA/DNA sequence or a oligo(dT) for annealing to a 3′ end of anyRNA with a poly(A) tail.

In one embodiment, the present invention provides a method of making adi-tagged DNA library, comprising the steps of: a) providing adouble-tagged oligonucleotide (DTO), sequentially comprising a sequencetag A, a linker, and a sequence tag B; b) providing a DNA or RNAfragment; c) connecting the DTO to the DNA fragment or a cDNA fragmentgenerated from the DNA or RNA fragment to form a circular DNA-DTOmolecule; d) generating a linear DNA from the circular DNA-DTO moleculewith sequence tag A and tag B at its ends. The collection of such linearDNAs with sequence tag A and tag B forms a di-tagged DNA library. TheDNA or RNA fragment refers to DNA or RNA sequences of certain size rangegenerated from a DNA or RNA sample. The DNA or RNA fragment can besingle stranded or double stranded. The double-stranded DNA is, undermost circumstances, blunt ended DNA. A cDNA from a DNA or RNA fragmentrefers a DNA sequence synthesized by DNA polymerization or reversetranscription using the DNA or RNA fragment as the template. A cDNAcomprises a DNA sequence complementary to its parent DNA or RNAsequence.

The DTO molecule can be a single stranded or a double stranded DNA. Whena single stranded RNA fragment is provided, a single stranded DTO isused as a primer and the RNA fragment is used as a template for areverse transcription to generate a cDNA fragment complementary to theparent RNA fragment. When a single stranded DNA fragment is provided, asingle stranded DTO is used as a primer and the DNA fragment is used asa template for a DNA polymerization reaction to generate a cDNA fragmentcomplementary to the parent DNA fragment. The DTO molecule is thusdirectly connected to the cDNA fragment via reverse transcription or DNApolymerization reaction. The single stranded DTO-cDNA sequence can beself-ligated to form a circular molecule. When a double stranded DNAfragment is provided, a double stranded DTO is used to ligate to twoends of the dsDNA fragment to form a circular molecule.

In some embodiment, the linear DNA fragments or free DTOs are digestedby exonucleases so that they can be easily separated from the circularDNA-DTO molecules. A single exonuclease (e.g. exonuclease V) or acombination of exonucleases (e.g. Lambda exonuclease/exonuclease I) areused to digest linear DNA with free double stranded or single strandedends. Exonuclease V has exonuclease activity towards ssDNA and dsDNA aswell as endonuclease activity towards ssDNA. It can be used to removelinear ssDNA and dsDNA from closed circular dsDNAs. If circular dsDNAshave nicks or gaps, exonuclease V should not be used to remove linearDNAs due to its ssDNA endonuclease activity. Exonucleases (e.g. Lambdaexonuclease) that cannot act upon nicked or gapped sites of a dsDNAshould be used instead. Lambda exonuclease catalyzes removal ofmononucleotides of dsDNAs from 5′ to 3′ direction, but cannot initiatedigestion at nicks or gaps of dsDNAs. Lambda exonuclease is a goodexonuclease to be used to digest linear dsDNA and free dsDTOs when anicked or gapped circular DNA is the desired product. Lambda exonucleasedigests one strand of a dsDNA, leaving a single stranded DNA, which canbe digested by a single stranded DNA specific exonuclease such asexonuclease I.

There are two methods for generating a linear DNA from a circularDNA-DTO molecule. If there is a breaking site within the DTO sequence,linear DNAs with sequence tag A and tag B can be generated by breakingat the breaking site. Alternatively, using a primer pair correspondingto sequence tag A and tag B, a PCR is performed to generate a di-taggedlinear DNA from the circular DNA-DTO molecule.

The linker of a DTO comprises a stopping sequence or a breaking site,wherein the stopping sequence comprises modified nucleotides or chemicalmoieties that can stop a DNA polymerization reaction, and the breakingsite comprises a special region of the DTO that is susceptible to photo,enzymatic or chemical cleavage.

In one embodiment, the invention provides a PCR-free method for making adi-tagged DNA sequence using a double stranded DTO. The method comprisesthe following steps: a, ligate two ends of a dsDNA fragment to adouble-stranded DTO with a breaking site to make a circular DNA-DTO; b,digest linear dsDNAs using a linear DNA specific exonuclease; c, use anappropriate method to cleave the circular DNA-DTO sequence at thebreaking site to generate a linear DNA sequence with two differentsequence tags at each end of the sequence. Conventional method foradding two sequence tags to DNA fragments involves randomly ligating twosequence tags to DNA fragments. This method results in ligation productswith only one sequence tag, two of the same sequence tag, and twodifferent sequence tags. A PCR is needed to selectively amplify theligation products with two sequence tags. The present invention directlyconnects the two ends of a DNA to two different sequence tags in a DTOmolecule. The advantage of this method is that no PCR amplification isrequired, therefore eliminating possible sequence bias caused by PCR.

For this method to be effective, it is very important to minimizeself-ligation within DTOs or DNA fragments, and increase the efficiencyof 1:1 (DTO: DNA fragment) ligation. In some embodiment, a singleAdenine nucleotide is added to the 3′ end of the dsDNA fragment using anon-proofreading polymerase (e.g. Taq DNA polymerase or Klenowfragments, 3′-5′ exo minus). A single 3′ T-overhang is added to thedouble-stranded DTO to minimize self-ligation. When ligating A-tailingDNA fragments and T-overhang DTOs, self-ligation within DNA fragments orDTOs can be minimized. Although using A/T tailing in DNA ligation isquite common in practice, ligation of DNAs with G/C tailing gives muchhigher ligation efficiency. A single Guanine nucleotide can be added toa blunt ended dsDNA using a non-proofreading polymerase such as Taq DNApolymerase. Incubating a blunt ended dsDNA with Taq DNA polymerase inthe presence dGTP only adds one single GMP to 3′ end of the dsDNA. Theaddition of dGMP to the end of dsDNAs is as efficient as addition ofdAMP by DNA polymerases (U.S. Pat. No. 7,723,103). DTOs with a singleC-overhang are then used to ligate with the DNAs with a singleG-tailing. Nucleotide tailing can also be added to the 3′ end of DNAfragments using terminal deoxynucleotidyltransferases (Promega, Madison,Wis.). Terminal deoxynucleotidyltransferases adds one or moretemplate-independent nucleotides to the 3′ terminal of DNA fragments,and complementary nucleotide overhangs should be added to DTOs. Sincethe number of nucleotides added by terminal deoxynucleotidyltransferasesto a DNA molecule is not fixed, during the ligation, a DNA ligase isadded together with a DNA polymerase that can extend 3′ end to fill gapsbetween the ends of DTOs and DNA fragments. Using G/C pair overhang cangreatly increase ligation efficiency between DTO and DNA fragments whileminimizing self-ligation within DTOs and DNA fragments themselves. Theratio between DTO and DNA fragments in the ligation reaction can bevaried to favor 1:1 (DTO:DNA fragment) ligation. This ratio (DTO: DNAfragment) can vary from 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, to 10:1,or higher.

After the ligation, an exonuclease (e.g. Exonulease V (NEB) or Plasmid-Safe™ DNase (Illumina)) or a combination of exonuclease (e.g. T7exonuclease/exonuclease I or Lambda exonuclease/exonuclease combination)that catalyzes hydrolysis of linear DNAs is added to remove linear DNAsincluding unligated DTOs, unligated DNA fragments, and linear ligationproducts. The digested products including mononucleotides and smalloligonucleotides, and exonuclease enzyme are removed from circularDNA-DTOs by DNA purification methods known to those skilled in the art.In another embodiment, a biotin moiety can be incorporated into DTOs andligated DTO-DNA products can be separated from unligated DNA fragmentsusing streptavidin beads.

The breaking site of the DTO may comprise, for example, asingle-stranded DNA/RNA, Uracil nucleotides, or other chemical moieties,which allows photo cleavage, enzymatic cleavage or chemical cleavage atthe breaking site. In some embodiment, DTOs have a single-stranded DNAor RNA sequence as the breaking site. A circular dsDNA-DTO is formedwith a stretch of single-stranded sequence between sequence tags A andB. S1 nuclease, an endonuclease active against single-stranded DNA orRNA sequences, can be used to cleave the singe-stranded region of thecircular DNA and generate a linear DNA sequence with tags A and B. Inanother embodiment, the breaking site is incorporated with uracilnucleotides on both strands. Uracil DNA Glycosylase (UDG) is used toremove a uracil base from the uracil nucleotide to form an abasic site(apurinic/apyramidinic site, also called AP site) while leaving thesugar-phosphate backbone intact. AP endonuclease (e.g. endonucleaseVIII) is then used to cleave DNA at the AP site and create a onenucleotide gap at the site of uracil nucleotide. When a uracil isincorporated in both strands of the breaking site in a double-strandedDTO, the combined treatment of UDG enzyme and AP endonuclease releases alinear DNA with sequence tags A and B at its ends. In anotherembodiment, a photo-cleavable nucleotide spacer is incorporated betweensequence tag A and tag B. A linear DNA is generated from the circularDNA-DTO molecular by cleaving the photo-cleavable spacer from thecircular molecule upon UV exposure. In another embodiment, nucleotideanalogs susceptible to chemical cleavage is incorporated into thebreaking site. For example, at least one of the nucleotide analogsselected from 5-hydroxy-dCTP, 5-hydroxy-dUTP, 7-Deaza-7-nitro-dATP, or7-Deaza-7-nitro-dGTP can be incorporated in each strand of the dsDTOmolecule. Chemical treatment of KMnO₄ and pyrrolidine cleaves thenucleotide analogs from the circular DNA-DTO molecule, generating alinear dsDNA with sequence tag A and tag B.

In some embodiment, PCR amplification is needed to increase the amountof di-tagged dsDNA output. The invention provides a method of making adi-tagged DNA comprises the steps of the following: a, ligate adouble-stranded DTO to DNA fragments to form a circular DNA-DTO product;b, use sequence tag A and B as PCR primers to amplify the DNA fragmentswith sequence tag A and B at both ends. In this method, it is desirableto have a stopping sequence to get a cleaner PCR product and preventformation of rolling cycle products. A stopping sequence, however, isnot required in this method. The DTO may comprise sequence tag A and Bwithout a linker sequence in between.

Mate pair DNA refers to a DNA sequence comprising two DNA segmentsoriginally located long distance apart, usually more than severalkilobases, in the genome. A mate pair library is comprised of mate pairDNA sequences with two different sequence tags attached at the ends. Inone embodiment, the present invention provides a simple and highlyefficient method for making mate pair DNA libraries. The methodcomprises the following steps: a, ligate a DTO to two ends of large DNAfragments to generate large circular DNAs; b, randomly fragment largecircular DNAs to small DNA fragments; c, perform end repair of the smallDNA fragments to make ready for ligation; d, self-ligate the small DNAfragments to generate small circular DNA; e, perform PCR with the smallcircular DNA to generate a linear DNA with tags A and B, which containssequences of both ends from a same large DNA fragment.

Ligation of a DTO to two ends of a large DNA fragment results in acircular DNA where both ends of the large DNA fragment are linked tosequence tags A and B in the DTO. In some embodiment, exonucleases areadded to remove linear dsDNA including unligated dsDTOs, linear DNAfragments and linear ligation products. Exonucleases that specificallydigest linear double stranded DNA, but have very little activity towardsclosed circle dsDNA can be used herein. Examples of such exonucleasesinclude exonuclease V and Plasmid-Safe™ DNase (an ATP-dependent DNaseavailable from IIlumina (San Diego, Calif.) that digests linear dsDNA,but not nicked or closed-circle dsDNA). The large circular DNAs are thenpurified from exonuclease enzymes and the digestion products using DNApurification and extraction kits from commercial sources such as Qiagen(Valencia, Calif.), Agilent Technology (Santa Clara, Calif.), andBeckman Coulter (Brea, Calif.). The large circular DNAs are randomlyfragmented to smaller DNA fragments using methods well known to personskilled in the art, including physical (e.g. sonication, physicalshearing, and nebulization) and enzymatic means (e.g. use of DNAse I orBenzonase). The small DNA fragments then undergo an end repair process,and are converted to be blunt ended with 5′-phosphate and 3′-OH, whichis ready for blunt end ligation. The end repair kits are available frommultiple commercial sources such as NEB, Invitrogen, and Illumina. Afterself-ligation of the small DNA fragments, those containing sequence tagsA and B are amplified by PCR reactions using primers corresponding tosequence tags A and B.

In some embodiment, exonucleases (e.g. exonuclease V or Plasmid-Safe™DNase) are added to digest linear unligated dsDNA. The small circularDNAs are then purified using DNA purification and extraction kits beforeproceeding to PCR amplification. In some embodiment, the DTO comprises abreaking site, and the small circular DNA is cleaved at the breakingsite to generate a linear DNA. PCR amplification is performed againstthe linear DNA to select DNA with two sequence tags. In some embodiment,the DTO comprises a stopping sequence, and PCR amplification isperformed directly on small circular DNAs to generate linear di-taggedDNAs. In another embodiment, modified nucleotide (e.g. biotinylatednucleotide) can be incorporated into DTO sequences. large and smallcircular DNA containing biotinylated DTO sequences can be isolated andenriched using streptavidin beads. Biotin-nucleotide can be incorporatedin the middle of DTO sequences, not at the ends. This helps to alleviatethe problem of low ligation efficiency caused by end biotin-nucleotides.

In some embodiment, the present invention provides a method of making amate pair DNA libraries from large DNA fragments using a nicked DTO. Themethod comprises the steps of the following: a, ligate both ends of alarge DNA fragments to a double-tagged oligonucleotide to generate alarge circular DNA, wherein the double-tagged oligonucleotide has a nicksite on each of the opposite strand; b, perform a nick translation toelongate the 3′ end of each nick site; c, break the large circular DNAat the nick sites to generate a smaller linear DNA with thedouble-tagged oligonucleotide; d, end repair the small linear DNA tomake it ligation ready; e. self-ligate the small linear DNA to form asmall circular DNA that contains end sequences of a large DNA fragmentand double-tagged oligonucleotide; f, perform PCR to amplify mate pairsequences between sequence tags A and B.

A nick in a dsDNA refers to a break of a phosphodiester bond of adjacentnucleotides in one strand. The nick sites can be introduced at anylocation along the DTO sequence, but not at the same location.Preferably, the nick site is positioned relatively close to the 3′ endof the DTO strand and both nicks are positioned about the same distanceaway from the 3′ termini. For example, the nick sites can be positioned6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or more nucleotides away fromthe 3′ end. Nick sites can be sealed by DNA ligase if both 5′ phosphateand 3′ hydroxyl group are available. To prevent nick sites from beingsealed during ligation, the 5′ phosphate group of both nick sites isreplaced with a 5′ hydroxyl group. To increase ligation efficiencybetween DTO molecules and large DNA fragments, a C or T-overhang isadded to the 3′ end of DTO molecules, and a complementary G or A-tailingis added to the blunt end of large DNA fragments using anon-proofreading DNA polymerase (e.g. Taq DNA ploymerase or Klenowfragment (exo-)).

After ligation, a nick translation is performed to extend the 3′ end ofthe nicks. A nick translation is elongating nucleotides from 3′ end of anick by a DNA polymerase, and at the same time removing nucleotides from5′−>3′ and replacing an old strand with a newly formed strand. DNApolymerases (e.g. E. coli DNA polymerase I) that have both DNApolymerase activity and 5′−>3′ exonuclease activity can be used for nicktranslation. The elongation distance is dependent on the processivity ofthe DNA polymerase used. By varying the amount of enzyme activity,incubation time and temperature, the number of bases added by a nicktranslation can be controlled. Preferably, the number of elongated basesfrom each nicked site is within the range of 50 bp to 300 bp so that theresulting di-tagged DNA is about 100 bp to 600 bp in length. Thedi-tagged DNA thus has about 50 bp to 300 bp sequences from each end ofthe parent DNA fragment. After nick translation, add an exonuclease with5′−>3′ activity (e.g. T7 exonuclease) to partially digest away thenicked strand and create a single-stranded gap region, which issubjected to cleavage by single-stranded DNA/RNA specific nuclease suchas S1 nuclease. Quickly inactivate exonuclease after digestion iscomplete, and use DNA purification methods (e.g. phenol chloroformextraction, or Agencourt AmPure DNA purification kit) to completelyremove the exonuclease. Residual nuclease activity could lead todegradation of final products. After the exonuclease is inactivated andremoved, S1 nuclease is added to make a cleavage at the single-strandedregion to release a smaller linear DNA fragment, namely a small linearDNA-DTO fragment, which contains two end sequences of an original largeDNA fragment linked by a DTO sequence. The small linear DNA fragmentsare end repaired to become blunt ended DNA. The blunt ended DNAs areincubated with T4 DNA ligase to form small circular DNAs. The unligatedlinear dsDNAs are removed by exonuclease treatment. PCR amplification isperformed on the small circular DNAs to select dsDNAs with sequence tagA and tag B at the ends.

In some embodiment, biotin-nucleotide can be incorporated into DTOsequences and biotinylated DNA-DTO fragments can be enriched by bindingto streptavidin-coated magnetic beads. The end repair and self-ligationcan be performed on the small linear DNA-DTO fragments bound tostreptavidin beads. Self-ligation of linear DNA-DTO sequence produces asmall circular DTO-DNA sequence that two end sequences of an originallarge DNA fragment are linked together with a DTO sequence. Using PCRprimers corresponding to sequence tags A and B, the small circularDNA-DTO products can be amplified by PCR to generate linear di-taggedmate pair DNAs. In some embodiment, the linker of DTO moleculescomprises a stopping sequence, which can lead to cleaner PCRamplification products. In some embodiment, the linker region of DTOmolecules comprises a breaking site, which provides a site for breakingthe small circular DNA-DTO molecule to generate a linear mate pair DNAwith sequence tags A and B at its ends. This linear mate pair DNA can befurther amplified by PCR.

In one embodiment, the invention provides a method for making adi-tagged dsDNA sequence from a RNA sequence. The method comprises thefollowing steps: a, annealing a DTO to the RNA sequence, wherein the DTOhas, from 5′ to 3′, a sequence tag A, a stopping sequence, a sequencetag B, and a priming sequence; b, performing a reverse transcription toextend the 3′ end of the DTO to make a cDNA-DTO molecule; c,self-ligating the 3′ and 5′ ends of the cDNA-DTO to generate a circularcDNA-DTO. The circularized cDNA-DTO is amplified by PCR to generate alinear dsDNA with sequence tag A and tag B. This method can be used forconverting a variety of RNAs such as total RNAs, mRNAs, rRNA depletedRNAs, and small RNAs into di-tagged dsDNAs.

In some embodiment of the present invention, the DTO comprises a mixtureof oligonucleotides with the priming sequence of each oligonucleotidehaving a random sequence, for example, a random hexamer. Random hexamerscan be primed to any complementary locations on a RNA sequence. Whenusing unfragmented RNAs as templates, the ratio of DTOs and RNAtemplates can be adjusted to favor production of small cDNA sequences.For example, using 250 ng hexamer primers per 5 μg of RNA can increaseyield of small cDNA products (<500 bp). To further favor generation ofsmall cDNA fragments, reverse transcriptases with less progressiveactivities could be chosen for the reverse transcription. An importantadvantage of this method is that it allows generation of small cDNAfragments from RNA molecules without fragmentation. Alternatively, RNAsamples can be first fragmented into desired sizes using methods thatare known to the people skilled in the art. For example, RNA can befragmented by heat or chemical treatments as described by Cloonan et al.(Nature Methods, 2008, 5: 613-619). The DTOs with random hexamers canthen be used to anneal to fragmented RNA templates and be extended byreverse transcriptases to make cDNAs. The 3′ and 5′ ends of a singlestranded cDNA-DTO are ligated using a ligase that can ligate singlestranded DNAs. T4 RNA ligase or CircLigase™ ssDNA ligase (Illumina, SanDiego, Calif.) can ligate single stranded DNA and can be used for thispurpose. CircLigase™ ssDNA ligase is a ligase that catalyzesintramolecular ligation (i.e. circularization) of ssDNA templates havinga 5′-phosphate and a 3′-hydroxyl group. The efficiency of self-ligationdepends on the length of cDNA sequences. The self-ligation based methodshould be most effective for converting small/micro RNAs to di-taggedsmall DNAs. This self-ligation based method also has advantages inworking with very small amount of start materials as the self-ligationefficiency is less affected by low concentrations of RNA/DNA molecules.

In some embodiments, a polyadenine (poly(A)) tail is added to a RNAfragment using a polynucleotide adenylyltransferase, also named poly(A)polymerase, and a poly(dT) tail is used as the priming sequence in theDTO. To anchor the DTO to the junction of poly(A) tail and non-poly(A)region of the RNA molecule, a joining sequence is added adjacent to the3′ end of the poly(dT) tail. The joining sequence has a general formulaof 5′-MN₁N₂ . . . N_(x)-3′, wherein M is any natural deoxyribonucleotideother than thymine nucleotide, N is any of four natural nucleotides,dAMP, dTMP, dCMP, dGMP, and x is an integer from 1 to 10, preferably 1to 3. When this method is applied to make library preparations fromfragmented RNAs, addition of a poly(A) tail to fragmented RNAs providesa priming site and ensures that cDNA synthesis starts from the 3′ end ofRNA fragments. For quantifying the amount of mRNA species using highthroughput sequencing, directly measuring the amount of RNA species witha poly(A) tail is sufficient. The DTOs with a poly(T) tail can bedirectly used to prime cDNA synthesis of poly(A)-RNAs. Addition of apolynucleotide other than a poly(A) tail to 3′ of RNA molecules can alsoserve the purpose of adding a priming site on RNA molecules. Forexample, a poly(U) polymerase (NEB, Ipswich, Mass.) can be used to add apoly(U) tail to RNAs. A person skilled in the art will understand thatthe present method of the invention can be similarly applied to RNAswith polynucleotide tails other than poly(A) tails.

Self-ligation of DTOs during the ligation step is an undesirable sideeffect that could become especially problematic when the amount ofstarting materials is very low. In some embodiment, a single strandednucleic acid specific nuclease is used to digest the un-annealed singlestranded DTOs. For example, exonuclease I, exonuclease T or Mung Beannuclease can be used for this purpose. In another embodiment of theinvention, a capture domain is added to RNA molecules and is used toseparate DTOs annealed to RNA molecules from the un-annealed ones. Acapture domain refers to a chemical structure or moiety incorporatedinto a nucleic acid sequence that allows the separation of the capturedomain containing nucleic acid sequence and any specifically boundnucleic acids from the rest of nucleic acid populations. For example, abiotin-ATP can be incorporated into RNA molecules using poly(A)polymerase and biotin-RNAs and the associated DTOs or cDNAs can becaptured by streptavidin or avidin-coated magnetic beads. The unboundDTOs can be washed away before ligation reaction. Biotin-ATP can beincorporated efficiently by the Poly (A) Polymerase. The tailing lengthand biotin density can be controlled by optimizing the reaction time,the ATP/biotin-ATP ratio and the total ATP concentration. In someembodiment, a ATP/biotin-ATP mixture is added during a polyadenylationreaction and biotin-AMPs are randomly incorporated into poly(A) tails.To ensure that biotin-AMPs are not incorporated adjacent to the 3′ endof RNA sequences, biotin-ATPs can be added at a later time of thepolyadenylation reaction. A biotin-(3′-deoxy)ATP can be added at the endof a polyadenylation reaction to add only one terminalbiotin-(3′-deoxy)AMP to poly(A) tails. Alternatively, biotinylated dNTPcan be incorporated into the cDNA during the reverse transcriptionreaction. Some Reverse transcriptase (e.g. M-MLV reverse transcriptase)can efficiently incorporate biotinylated dNTP into cDNA sequences. Theprocedure to incorporate biotinylated dNTP into cDNA sequences is wellknown to those skilled in the art and is well described in technicalpublications, for example, technical articles from TrilinkBiotechnologies (San Diego, Calif.). The biotin-cDNA can then beseparated from unbound DTOs using Streptavidin coated magnetic beads.

The invention provides a method for making a di-tagged dsDNA from asingle stranded DNA sequence using a single stranded DTO, wherein theDTO has, from 5′ to 3′, a sequence tag A, a stopping sequence, asequence tag B, and a priming sequence. The method comprises thefollowing steps: a, annealing the ssDTO to the ssDNA sequence; b,extending the 3′ end of the DTO using a DNA polymerase to make acDNA-DTO; c, ligating the 3′ and 5′ ends of the cDNA-DTO to form acircular cDNA-DTO molecule. The circularized cDNA-DTO can be amplifiedby PCR to generate a linear dsDNA with sequence tags A and B. In oneembodiment, the priming sequence of the DTO comprises a random hexamer.In another embodiment, a poly(dA) tail is added to the 3′ end of DNAmolecules by a terminal transferase and a DTO with a poly(dT) is used asa primer for synthesizing a cDNA. Biotinylated dAMPs is incorporatedinto the poly(dA) tail of DNA molecules using a terminal transferase.The biotin-poly(dA) tail can be used to separate cDNA/biotin-DNA hybridsfrom unbound DTOs. In another embodiment, a single stranded nucleic acidspecific nuclease (e.g. exonuclease I, exonculease T, or Mung Beannuclease) is used to digest single stranded DTO molecules.

Preparation of single-stranded DNA is useful in many applications, suchas DNA methylation assays or used as capturing probes. The presentinvention provides a method of making a di-tagged single-stranded DNAfrom a double-stranded DNA. The method comprises the steps of thefollowing: a, ligate a double-stranded DNA to a double-taggedoligonucleotide to generate a circular dsDNA, wherein the double-taggedoligonucleotide sequentially comprises a sequence tag A, a breakingsite, a sequence tag B, and wherein the double-tagged oligonucleotidehas a nick or a gap; b, remove the nicked/gapped strand using anexonuclease to generate a circular single-stranded DNA; c, break thecircular single-stranded DNA at the breaking site to generate a lineardi-tagged single-stranded DNA.

A double-stranded DTO with at least one single nick or a gap in one ofthe two strands is used in this method. The 5′ end of the nick is ahydroxyl group so that it cannot be sealed during ligation. Thedouble-tagged oligonucleotide has either a blunt end or a C- orT-overhang. The breaking site of the DTO comprises a Uracil-nucleotideon the un-nicked/un-gapped strand. Ligate a DTO sequence with onenick/gap to a dsDNA to generate a circular dsDNA with a nick or a gap.To remove the nicked or gapped strand of the circular dsDNA, incubatethe circular dsDNA with an exonuclease that is able to progressivelycleave nucleotides from a nick site or a gap of a dsDNA. Examples ofsuch exonucleases include T7 exonuclease, which can initiate aprogressive cleavage from 5′−>3′, and exonuclease III, which can removenucleotides from 3′−>5′. After digestion is complete, quickly inactivateexonucleases and use DNA purification and extraction methods (e.g.phenol chloroform extraction, cesium chloride centrifugation orAgencourt AmPure beads-based DNA purification methods) to removeexonucleases from the single-stranded circular DNA. Residual enzymeactivity could lead to degradation of desired DNA products. After theexonuclease is removed, the circular ssDNA can be cleaved at thebreaking site of Uracil-nucleotide when incubating with UDG enzyme andan AP endonuclease such as endonuclease VIII, therefore generating alinear single-stranded DNA with sequence tag A and B at its ends. Insome embodiment, the breaking site of the DTO comprises a singlestranded RNA, which can be cleaved by RNase treatment.

In another embodiment, the double-tagged oligonucleotide comprises astopping sequence. Since nucleotide analogs or other non-nucleotidechemical moieties constituted of stopping sequence cannot form normalbase pairing under most circumstances, a stopping sequence in a doublestranded DTO is likely to create a unconnected gap. For example, onestrand of the double stranded DTO may comprise a stretch of abasicnucleotides linking sequence tag A and tag B, and the other strandcomprises a sequence tag A and tag B unconnected. In some embodiment,the stopping sequence comprises a single stranded RNA or a doublestranded RNA. Most DNA polymerases either cannot use RNA as template ordoes so at very low efficiency. Incorporating multiple ribonucleotidesinto a DTO sequence can effectively prevent extending DNA polymerizationacross the RNA region. After the DTO and the dsDNA sequence are ligatedto form a circular DNA-DTO, using one primer annealing to eithersequence tag A or B, single-stranded DNA can be amplified by a linearPCR. The stopping sequence ensures the generation of single-strandedDNAs with two sequence tags at the ends and prevents formation ofrolling cycle products by PCR amplification.

EXAMPLES Example 1 A PCR-Free Method For Making a Di-Tagged DNA Library

Conventional methods randomly ligating two sequence tags to DNAfragments result in ligation products with only one tag, two of the sametag, and two different tags at ends of the DNA sequences. A PCR isneeded to selectively amplify the ligation products with two sequencetags. This example (FIG. 2A) shows how to make di-tagged DNA librariesfrom DNA fragments using a double-tagged oligonucleotide. This methoddoes not need PCR amplification or selection. The starting material isblunt ended DNA fragments. A DTO sequence having a sequence tag A, abreaking site, and a sequence B is used in this example. The breakingsite of the DTO sequence has at least one Uracil nucleotide in each oftwo strands.

G-Tailing of DNA Fragments

Ligation efficiency between blunt end or A/T pair nucleotides is usuallyquite low. In order to increase ligation efficiency, Guanine nucleotidesare added to 3′ OH terminus of DNA fragments using terminaldeoxynucleotidyltransferase. Terminal deoxynucleotidyltransferase is atemplate-independent DNA polymerase that catalyzes repetitive additionof deoxyribonucleotides to 3′ OH of a DNA molecule. Incubate blunt endedDNA fragments with a terminal deoxynucleotidyl transferase (Illumina,San Diego, Calif.) and dGTP to add Guanine nucleotides to the 3′ end ofDNA fragments. By varying enzyme concentration, incubation time, anddGTP to DNA fragment ratio, the reaction can be controlled so that oneto five Guanine nucleotides are added to the termini of DNA fragments.To be complementary to the 3′ G overhang of DNA fragments, A stretch offive Cytosine nucleotides 3′ overhang is added to the DTO sequence.

DNA Ligation and Circularization

Incubate DNA fragments with 3′ G overhang, DTOs with 3′ C overhang, T4DNA ligase, and a DNA polymerase I, large fragment (exo-), whichmaintains 5′−>3′ DNA polymerase activity, but lacks 3′−>5′ and 5′−>3′exonuclease activity in the presence of dGTP and dCTP. 3′ C overhang ofDTO sequences anneals to 3′ G overhang of DNA fragments. The DNApolymerase I, large fragment (exo-) adds G or C nucleotides to fill inany gap between the annealing strands. T4 DNA ligase further ligates twostrands to form a circular product where both ends of DNA fragments arelinked to sequence tags A and B of DTO molecule. Incubate the ligationreaction mixture with exonuclease V which catalyzes digestion ofunligated DNA fragments, unligated DTOs, and linear ligation products.The circular DNA-DTO ligation products are purified with AMPure XP beads(Beckman Coulter, Brea, Calif.) to remove free dNTP, free DTO andenzymes.

Generation of Linear DNA Library

The purified circular DNA-DTO product is linearized by incubating withUracil DNA Glycosylase (UDG) and endonuclease VIII according to theprotocol of NEB (Ispwich, Mass.). The combination of UDG andendonuclease VIII generates a break at the position of Uracil nucleotideand releases a linear DNA with sequence tags A and B at its ends.

Example 2 Method For Making a Di-Tagged DNA Library Using a Gapped DTO

This example illustrates a method of making di-tagged DNA library fromdsDNA fragments using a gapped dsDTO. The DTO has a sequence tag A, asingle stranded gap (as a breaking site), sequence tag B, and a 3′ dToverhang (see FIG. 2B).

A-Tailing of DNA Fragments and Ligation of DNA Fragments & DTOs

The blunt ended DNA fragments are incubated with Taq DNA polymerase inthe presence of only dATP and a single dA is added to the 3′ terminus ofDNA fragments. DNA fragments with an A-overhang are ligated to DTO witha dT-overhang to form a circular DNA-DTO molecule. The circular DNA-DTOmolecule has a single stranded gap with free 3′ and 5′ termini on thegapped strand. Lambda exonuclease that catalyzes removal of nucleotidesfrom 5′ to 3′, but cannot act on nicks or gaps of a dsDNA, is used toremove unligated dsDNA, free dsDTO, and linear ligation products whileleaving the circular ligation products intact. Exonuclease I is used tofurther digest the single stranded DNA resulted from Lambda exonucleasedigestion. After digestion, exonucleases are denatured and removed byphenol chloroform extraction and ethanol precipitation.

Generation of Linear Di-Tagged DNA

The purified circular DNA-DTOs are treated with S1 nuclease to generatelinear di-tagged DNAs. If needed, further PCR amplification can beperformed to amplify the di-tagged DNA.

Example 3 Method For Making a Mate Pair DNA Library Using aDouble-Stranded DTO

This example shows a method of using a double-stranded DTO to make adi-tagged mate pair DNA library containing two DNA segments that areseparated by several kilobases in the genome (FIG. 3). A DTO sequencehaving a sequence tag A, a uracil nucleotide on each of its two strand(as a breaking site), and a sequence tag B is used in this method. A 3′dC overhang is added to the DTO sequences.

Ligation of DTO and Large DNA Fragments

Genomic DNA is fragmented into large DNA fragments of 2-3 kb in size,and is end-repaired to generate blunted end fragments having 5′phosphate and 3′ OH. The large DNA fragments are incubated with Taq DNApolymerase in the presence of dGTP only. Taq DNA polymerase adds onesingle 3′ GMP overhang to blunt ended dsDNAs. The two ends of large DNAfragment with a single dG tail are ligated to a DTO with a dC overhangto form a large circular DNA-DTO molecule using T4 DNA ligases. Theunligated linear DNA sequences, unligated DTOs, and linear ligationproducts are digested by exonuclease V. The large circular DNA-DTOmolecules are purified using AMPure XP beads, with enzymes, shortoligonucleotides, and mononucleotides being removed.

Random Fragmentation to Self-Ligation to Generate Circular Mate Pair DNA

The large circular DNA-DTO molecules are randomly fragmented intosmaller fragments, for example, 200 to 400 bp in size, usingnebulization. The small DNA fragments are end repaired to generate bluntended DNA using NEB End repair module. After end repair, the small DNAfragments are incubated with T4 DNA ligase to form self-ligationproducts. The circular self-ligation DNA with DTO sequences comprisesmate pair DNAs having sequences from both ends of the original large DNAfragments. The linear DNAs are removed by exonuclease V treatment andsmall circular DNAs are purified using AMPure XP beads.

PCR Amplification to Generate Linear Mate Pair DNA

Treat the small circular DNAs with UDG and endonuclease VIII to generatea linear DNA with sequence tag A and tag B. Perform 5 to 15 cycles ofPCR to amplify DNAs between sequence tag A and tag B. If needed, sizeselection and purification of linear mate pair

DNAs can be performed on an agarose gel.

Example 4 Method for Making a Mate Pair DNA Library Using a Nicked dsDTO

This example shows a method of using a nicked DTO to make a di-taggedmate pair DNA library (FIG. 3). The nicked DTO sequence used in thisexample has a nick in each of the two strands, wherein the nick has a 5′and 3′ hydroxyl group so that the nick cannot be sealed by a DNA ligase.The nicked DTO has a sequence tag A, a uracil nucleotide on each of itstwo strand, a sequence tag B, and a 3′dC overhang.

Ligation of DTOs and Large DNA Fragments

ligation of nicked DTO sequences to large DNA fragments is proceeded asdescribed in Example 4. Since the nick has a 5′ and 3′ OH group, thenick cannot be sealed during the ligation and the nick is kept intactafter the ligation.

Nick Translation and Self-Ligation to Generate Circular Mate Pair DNAs

The purified large circular DNA-DTO molecule is incubated with E. coliDNA polymerase I and a dNTP mixture. The DNA polymerase I elongates the3′ terminus of the nick, removes nucleotides from 5′−>3′, replaces themwith dNTP and shifts the “nick” away from the original location. Thedistance that the nick moves along a DNA strand depends on theprocessivity of the DNA polymerase I. The amount of DNA polymerase I,incubation temperature, and incubation time can be optimized to extend100 to 200 nucleotides from each nick site. Stop nick translation byinactivating DNA polymerase I and remove the enzyme and free dNTP fromthe nicked DNA using AMPure XP beads. Add T7 exonuclease for controlledtime period to generate a gap at the nick site. Use S1 nuclease to breakthe large circular DNA-DTO at the newly formed gap position to generatesmall DNA fragments. The small DNA fragments are end repaired togenerate blunt ended DNA using a NEB End repair module. After endrepair, the small DNA fragments are incubated with T4 DNA ligase to formself-ligation products. The small circular DNA treated with exonucleaseV and are further purified using AMPure XP beads.

PCR Amplification to Generate Linear Mate Pair DNA

Incubate the small circular DNAs with UDG and endonuclease VIII togenerate a break at the location of a uracil nucleotide. Perform 5 to 15cycles of PCR to amplify DNAs between sequence tags A and B. If needed,size selection and purification of linear mate pair DNAs can beperformed on an agarose gel.

Example 5 Method For Making a Di-Tagged DNA Library From a RNA SampleUsing Random Hexamers as a Priming Sequence

The method of the invention can be applied to make double-tagged DNAlibraries from a RNA or DNA sample. The double-tagged DNA libraries canbe used for cloning into a vector, generating hybridization probes, invitro transcription, high throughput sequencing, and other applications.This example illustrates the procedure for making di-tagged DNAlibraries from a RNA sample suitable for high throughput sequencing(FIG. 5).

Starting Materials

The starting materials for making a di-tagged DNA library can be totalRNA, mRNA, rRNA depleted RNA, and small RNA isolated from cells ortissues. The method of the invention is especially effective forpreparing di-tagged DNA libraries from small amounts of RNA samples whenthe resources are scarce such as RNA samples extracted from thoseassociated with RNA binding proteins in CLIP assays, RNA samplesisolated from small quantities of cells, or RNA samples purified frombiopsies.

When using random hexamers as the priming sequence, it is possible todirectly use full length RNAs as templates to generate cDNA fragmentswith size distributions (e.g. 200 to 500 bp) suitable for highthroughput sequencing. Random hexamer primers can anneal to anycomplementary location on the full length RNA and initiate a cDNAsynthesis. Using high primer to RNA ratio (e.g. 250 ng random hexamerper 5 μg RNA), the production of small size cDNA (<500 bp) by reversetranscription is favored. Alternatively, full length RNA molecules arefragmented into desired sizes using methods well known in the art. Thefragmented and purified RNA can then be used as starting materials formaking di-tagged DNA libraries.

Reverse Transcription to Synthesize cDNAs

A single-stranded DTO having, from 5′ to 3′, a sequence tag A, a stretchof five tetrahydrofuran abasic nucleotides, a sequence tag B, and arandom hexamer is used as a primer and purified RNAs (fragmented orunfragmented) are used as templates for cDNA synthesis according to areverse transcription protocol from New England BioLabs (Ipswich,Mass.). Briefly, 10 μM DTOs mixed with 50 to 200 ng mRNA and a dNTPmixture (1 mM of each dNTP) in RNAse-free water is heated for 3 to 5minutes at temperature 65 to 75° C. to denature the RNA. Briefly spindown the RNA/DTO solution and promptly put it in ice. Add RNaseinhibitor, a reverse transcriptase, and 10× reverse transcription bufferto the RNA/DTO solution and incubate for 45 to 60 minutes at recommendedtemperatures to synthesis cDNAs. After the synthesis of cDNA sequences,cDNA-DTOs are purified using AMPure XP beads (Beckman Coulter, Brea,Calif.) to remove enzymes, free NTPs and free DTO (<100 bp). RNAtemplates are later removed by RNase A and RNase H incubation. Thesingle-stranded cDNAs are purified using Zymo DNA clean and concentratorkit (Zymo Research Corporation, Irvine, Calif.).

Self-Ligation to Add a 3′ End Sequence Tag

Purified single stranded cDNAs with DTO at its 5′ end are self-ligatedusing CircLigase™ (Illumina, San Diego, Calif.) according tomanufacturer's instruction. The resulting circularized cDNA has sequencetags A and B at its 3′ and 5′ ends, respectively.

PCR Amplification to Generate dsDNAs With Two Sequence Tags

The circularized cDNAs are purified and recovered using a Zymo DNA cleanand concentration kit. Using primers corresponding to sequence tags Aand B, the circularized cDNAs are amplified to make linear double-taggeddsDNAs. The stopping sequence between the sequence tags A and B lacksbase-pairing ability with any natural nucleobases and is used to preventDNA polymerase from extending nucleotide polymerization beyond sequencetag A or B. The stopping sequence prevents rolling cycle amplificationduring PCR and leads to a linear tagged dsDNA with a clean background.

Example 6 Method For Making a Di-Tagged DNA Library From a RNA SampleUsing Poly(dT) as a Priming Sequence

The method of the invention can be applied to make a di-tagged DNAlibrary from small RNA fragments using a poly(dT) tail as the primingsequence of a DTO. Using six random hexamer as a priming sequence, cDNAsynthesis can be started in any location of a RNA template. Usingpoly(dT) as the priming sequence in the DTO ensures that the 3′ endsequence of a RNA template is included in the cDNA sequence. This methodis applied to small RNA sequences that have already fragmented,purified, and end repaired. It can be used for deep sequencing of wholetranscriptome or quantitating mRNA expression. The poly(dT) tailcontains at least 10, preferably 15 to 20, dTMPs in its sequence.Compared to random hexamer priming, priming with poly(dT) tail providesa higher priming specificity and efficiency (FIG. 6).

If quantitating of RNA expression is needed, RNA fragments do not needto be added a poly (A) tail. Only RNA fragments with a natural poly(A)tail will be reverse transcribed into cDNAs. If deep sequencing isneeded, purified RNA fragments are incubated with a poly(A) polymerasein the presence of ATP and biotin-ATP according to manufacturer'sinstruction (NEB, Ipswich, Mass.). The tailing length and biotin densitycan be controlled by optimizing the reaction time, the ATP/biotin-ATPratio and the total ATP concentration. The biotinylated poly(A)-RNAs arereverse transcribed to cDNAs using poly(dT) of DTOs as primers. Thebiotin-RNA/cDNA hybrids are pulled down by streptavidin-coated magneticbeads and the streptavidin beads are washed three times to removeunbound DTOs. Subsequent RNase treatment, self-ligation, and PCRamplification are proceeded as described above.

Example 7 Method For Making a Single-Stranded DNA From Double-StrandedDNA

This example shows a method of using a nicked DTO to make a di-taggedsingle-stranded DNA from a dsDNA (FIG. 9). The nicked DTO sequence usedin this example has only one nick in one of the two strands, wherein thenick has a 5′ and 3′ hydroxyl group so that the nick cannot be sealed bya DNA ligase. The nicked dsDTO has a sequence tag A, a uracil nucleotideon the un-nicked strand (as a breaking site), a sequence tag B, and a 3′dC overhang.

Ligation of Nicked DTO and dsDNA

Ligation of nicked DTO sequences to large DNA fragments to form a nickedcircular DNA-DTO molecule is proceeded as described in Example 3. Sincethe nick has a 5′ and 3′ OH group, the nick cannot be sealed during theligation and the nick is kept intact after the ligation.

Removal of One DNA Strand Using T7 Exonuclease

Incubate T7 exonuclease and exonuclease I with the circular dsDNA-DTO toremove the nicked strand of the circular DNA-DTO and other lineardsDNAs. T7 exonuclease initiates nucleotide removal at nicked site from5′−>3′, and it removes mononucleotides from a dsDNA with both 5′phosporylated and unphosphorylated end. T7 exonuclease also degrades onestrand of linear dsDTO and dsDNA from 5′−>3′, leaving undigestedsingle-stranded DNAs that can be further digested by exonuclease I.After the exonuclease digestion, denature and remove T7 exonuclease andexonuclease I using phenol chloroform extraction and ethanolprecipitation.

Generation of Linear Single-Stranded DNA With Two Tags

The purified circular single-stranded DNA-DTO product is linearized byincubating with UDG and endonuclease VIII according to the protocol ofNEB. The combination of UDG and endonuclease VIII generates a break atthe position of Uracil nucleotide and releases a linear DNA withsequence tags A and B at its ends. Further PCR amplification can beperformed if needed.

While the present invention has been described in some detail forpurposes of clarity and understanding, one skilled in the art willappreciate that various changes in form and detail can be made withoutdeparting from the true scope of the invention. All figures, tables,appendices, patents, patent applications and publications, referred toabove, are hereby incorporated by reference.

1. A method of making a di-tagged DNA library, comprising the steps of:a) providing a double-tagged oligonucleotide (DTO), sequentiallycomprising a sequence tag A, a linker, and a sequence tag B; b)providing a DNA or RNA fragment; c) connecting said DTO to said DNAfragment or a cDNA fragment complementary to said DNA or RNA fragment toform a circular DNA-DTO molecule; d) generating a linear DNA from saidcircular DNA-DTO molecule, having sequence tag A and tag B at its ends,therefore obtaining a di-tagged DNA library.
 2. The method of claim 1,wherein said linker comprises a stopping sequence or a breaking site. 3.The method of claim 2, wherein said stopping sequence comprises modifiednucleotides or chemical moieties that can stop a DNA polymerizationreaction.
 4. The method of claim 3, wherein said stopping sequencecomprises abasic nucleotides.
 5. The method of claim 3, wherein saidstopping sequence comprises nucleotide analogs that are sterically orelectronically incompatible with active sites of DNA polymerases.
 6. Themethod of claim 3, wherein said stopping sequence comprises nucleosideanalogs selected from 4-methylidole β-nucleoside, α-naphthalenenucleoside, and α-pyrene nucleoside.
 7. The method of claim 3, whereinsaid chemical moiety comprises DNA spacers selected from C3phosphoramidite spacer, C9 triethylene glycol spacer, or C18hexa-ethyleneglycol spacer.
 8. The method of claim 2, wherein saidbreaking site comprises a special region of said DTO that is susceptibleto photo, enzymatic or chemical cleavage.
 9. The method of claim 8,wherein said breaking site comprises uracil nucleotides.
 10. The methodof claim 8, wherein said breaking site comprises single stranded DNA orRNA.
 11. The method of claim 8, wherein said breaking site comprises aphoto-cleavable nucleotide spacer.
 12. The method of claim 8, whereinsaid breaking site comprises modified nucleotides selected from5-hydroxy-dCTP, 5-hydroxy-dUTP, 7-Deaza-7-nitro-dATP, and7-Deaza-7-nitro-dGTP.
 13. The method of claim 1, wherein means ofconnecting said DTO and said DNA fragment or said cDNA is selected fromDNA ligation, reverse transcription, or DNA polymerization.
 14. Themethod of claim 1, after step c), further comprising isolating circularDNA-DTO molecule by removing linear DNAs with exonuclease treatment. 15.The method of claim 1, wherein means of generating said linear DNA fromsaid circular DNA-DTO molecule is selected from PCR amplification andbreaking at said breaking site.
 16. The method of claim 1, wherein saidDTO is a double stranded DNA and a double stranded DNA fragment isprovided in step b).
 17. The method of claim 16, wherein said linker ofsaid DTO comprises a breaking site.
 18. The method of claim 17,comprising the steps of: I. connecting said DTO to said DNA fragment toform said circular DNA-DTO molecule by ligation; II. digesting linearDNAs using exonucleases; III. generating said linear DNA with sequencetag A and tag B from said circular DNA-DTO molecule by breaking at saidbreaking site.
 19. The method of claim 18, wherein said DNA fragmentcomprises 3′ overhang of a stretch of same nucleotides, and said DTOcomprises 3′ overhang of a stretch of complementary nucleotides.
 20. Themethod of claim 18, wherein said DNA fragment comprises a single 3′A-overhang, and said DTO comprises a single 3′ T-overhang.
 21. Themethod of claim 18, wherein said DNA fragment comprises a single 3′G-overhang, and said DTO comprises a single 3′ C-overhang.
 22. Themethod of claim 18, wherein said breaking site comprises uracilnucleotides; and wherein said breaking site is broken by treatment ofuracil DNA glycosylase and endonuclease VIII.
 23. The method of claim18, wherein said breaking site comprises a single stranded DNA/RNAsequence; and wherein said breaking site is broken by treatment ofnuclease S1.
 24. The method of claim 16, comprising the steps of I.connecting said dsDTO with said dsDNA to form a circular DNA-DTOmolecule by ligation; II. digesting linear DNA with exonucleasetreatment; III. randomly fragmenting said circular DNA-DTO molecule intosmaller sequences, IV. performing end repair and self-ligation of saidsmaller sequences to form a smaller circular DNA;
 25. The method ofclaim 24, wherein said DTO comprises a stopping sequence,
 26. The methodof claim 25, wherein said stopping sequence comprises abasicnucleotides.
 27. The method of claim 24, further comprising PCRamplification with said smaller circular DNA to generate a linear DNAwith sequence tag A and tag B.
 28. The method of claim 24, wherein saidlinker of said DTO comprises a breaking site.
 29. The method of claim28, further comprising removing linear DNA by exonuclease treatment,inactivating and removing exonuclease, and breaking said smallercircular DNA at the breaking site to generate a linear DNA with sequencetag A and tag B.
 30. The method of claim 24, wherein said DTO comprisesa capture domain.
 31. The method of claim 1, wherein said DTO is adouble-stranded DNA with a nick on each of its strands and a doublestranded DNA fragment is provided in step b).
 32. The method of claim31, comprising the steps of: I. connecting said DTO to said DNA fragmentto form a large circular DNA-DTO by ligation; II. performing nicktranslation to elongate 3′ end of each said nick site; III. using T7exonuclease to create a single stranded DNA gap and S1 nuclease tocleave said single stranded DNA gap; IV. performing end repair andself-ligation to form a smaller circular DNA-DTO;
 33. The method ofclaim 32, wherein said DTO comprises a breaking site.
 34. The method ofclaim 33, further comprising breaking said smaller circular DNA at saidbreaking site to generate a linear DNA with sequence tag A and tag B.35. The method of claim 34, further comprising PCR amplification of saidlinear DNA with sequence tag A and tag B.
 36. The method of claim 32,wherein said DTO comprises a stopping sequence.
 37. The method of claim36, wherein said stopping sequence comprises abasic nucleotides.
 38. Themethod of claim 36, further comprising PCR amplification of said smallercircular DNA-DTO to generate a linear DNA with sequence tag A and tag B.39. The method of claim 1, wherein a single stranded RNA or DNA fragmentis provided in step b), and said DTO comprises a single-stranded DNA, astopping sequence, and a priming sequence.
 40. The method of claim 39,comprising the steps of: a) synthesizing a cDNA-DTO using said DTO as aprimer and said RNA or DNA fragment as a template; b) ligating 3′ and 5′end of cDNA-DTO to form a circular DNA-DTO molecule; c) performing a PCRamplification to generate a linear DNA with sequence tag A and tag B atits ends.
 41. The method of claim 40, wherein said priming sequencecomprises a random sequence.
 42. The method of claim 40, said primingsequence comprises a poly(dT) tail.
 43. The method of claim 40, whereina poly(A) tail is added to said RNA fragment.
 44. The method of claim40, wherein said poly(A) tail is incorporated with at least onebiotin-AMP.
 45. The method of claim 40, wherein said stopping sequencecomprises abasic nucleotides.
 46. A method of making a di-taggedsingle-stranded DNA from a double-stranded DNA, said method comprisingthe steps of: e) providing a double-stranded double-taggedoligonucleotide sequentially comprises a sequence tag A, a breakingsite, sequence tag B, and a single nick or gap in one of the twostrands; f) ligating two ends of said double-stranded DNA to saiddouble-tagged oligonucleotide to generate a circular DNA-DTO; g)removing the nicked/gapped strand using an exonuclease to generate acircular single-stranded DNA; h) breaking said circular single-strandedDNA at said breaking site to generate a linear di-tagged single-strandedDNA.
 47. The method of claim 46, wherein said exonuclease comprises T7exonuclease and exonuclease I.
 48. The method of claim 46, wherein saidbreaking site comprises a uracil nucleotide.
 49. The method of claim 46,further comprising performing a single primer PCR to amplify saiddi-tagged single-stranded DNA.